The Three Methyl-CpG-binding Domains of AtMBD7 Control Its Subnuclear Localization and Mobility

Abstract

Three methyl-CpG-binding domain (MBD) proteins in Arabidopsis, AtMBD5, AtMBD6, and AtMBD7, are functional in binding methylated CpG dinucleotides in vitro and localize to the highly CpG-methylated chromocenters in vivo. These proteins differ, however, in their subnuclear localization pattern; AtMBD5 and AtMBD6, each containing a single MBD motif, show preference for two perinucleolar chromocenters, whereas AtMBD7, a naturally occurring poly-MBD protein containing three MBD motifs, localizes to all chromocenters. Here we studied the significance of multiple MBD motifs for subnuclear localization and mobility in living cells. We found that the number of MBD motifs determines the subnuclear localization of the MBD protein. Furthermore, live kinetic experiments showed that AtMBD7-green fluorescent protein (GFP) has lower mobility than AtMBD5-GFP and AtMBD6-GFP, which is conferred by cooperative activity of its three MBD motifs. Thus, the number of MBD motifs appears to affect not only binding affinity and mobility within the nucleus, but also the subnuclear localization of the protein. Our results suggest that poly-MBD proteins can directly affect chromatin structure by inducing intra-and interchromatin compaction via bridging over multiple methylated CpG sites.Cytosine methylation is a common epigenetic modification of most eukaryotic nuclear DNA. Intensive studies demonstrated the central role played by cytosine methylation in genome organization, gene expression, and growth and development. The way by which the methyl group is interpreted into a functional state has begun to be unraveled with the isolation and characterization of methylated DNA-binding proteins. This group of proteins includes an evolutionary conserved protein family, initially described in animals, termed methyl-CpGbinding domain (MBD) 2 proteins (1). Biochemical and molecular analyses suggest that mammalian MBDs induce the formation of a repressive chromatin structure at methylated CpG (mCpG) sites by the recruitment of various chromatin factors including histone deacetylases, histone methyltransferases, and ATPase-dependent nucleosome remodeling factors (2, 3). Thus far, of the 13 AtMBD proteins found in Arabidopsis, only AtMBD5, AtMBD6, and AtMBD7 were found to bind mCpG sites in vitro and to localize in vivo to the highly methylated chromocenters (ChCs) (reviewed in Refs. 4 and 5). This chromocenter localization is CpG methylation-dependent inasmuch as it was disrupted in the hypomethylated mutants ddm1 and met1 (6). Yet, these three AtMBDs display different localization patterns within Arabidopsis nuclei; AtMBD7 is predominantly localized at all ChCs, whereas AtMBD5 and AtMBD6 show preference for two perinucleolar ChCs adjacent to the ribosomal DNA clusters (6). A major structural feature distinguishing AtMBD7 from AtMBD5 and AtMBD6 is the presence of three MBD motifs within its protein sequence. Notably, naturally occurring poly-MBD proteins appear to be unique to plants as to date no such protein has been found in animals. Here we studied...

Methyl-CpG-Binding Domain Protein MBD7 Is Required for Active DNA Demethylation in Arabidopsis

“…Our study indicates that MBD7 prefers to target the genomic regions with high density of DNA methylation around chromocenters, which is consistent with previous studies showing that MBD7 is intensively localized in the chromocenters (Zemach et al, 2008). The mutations in met1 and ddm1 abolish the recruitment of MBD7 to chromocenters (Zemach et al, 2005).…”

“…MBD7 is a methyl-CpG-binding domain (MBD) protein containing three MBD motifs that bind in vitro to methylated symmetric CG sites. MBD7 localizes to all highly CpG-methylated chromocenters in vivo (Zemach and Grafi, 2003;Zemach et al, 2008). Recruitment of MBD7 to chromocenters is disrupted in decrease in DNA methylation1 (ddm1) and met1, two mutants with great reductions in DNA methylation, suggesting that DNA methylation is required for proper MBD7 localization (Zemach et al, 2005).…”

Regulation of Active DNA Demethylation by a Methyl-CpG-Binding Domain Protein in Arabidopsis thaliana

“…while, in DNA hypomethylation mutants ddm1 and met1 they dissociate from chromocenters, indicating their methyl-CpG-binding activity in vivo [ 18 ]. Different from AtMBD7, AtMBD5 and AtMBD6 mainly localize to chromatin regions covered by ribosomal DNA (rDNA) gene clusters [ 18 , 19 ]. Snf2 family nucleosome remodeler DDM1 is able to bind AtMBD5 and AtMBD6 in vitro and facilitate their heterochromatin localization [ 18 ].…”

Tomato MBD5, a methyl CpG binding domain protein, physically interacting with UV‐damaged DNA binding protein‐1, functions in multiple processes

“…a,b). Because it is previously reported that the Arabidopsis homologs of SlMBD5, AtMBD5 and AtMBD6 could form both homo‐ and heterodimers through their MBD motifs (Zemach et al ., ), we hypothesized that the trimmed SlMBD5 might associate with DDB1‐CUL4 via dimerization of its MBD with the endogenous tobacco MBD homolog of SlMBD5. To verify this, we first detected SlMBD5 could be dimerized by yeast two‐hybrid assay (Fig.…”